In the previous years we have shown that RNase-(-118) lacking 6 carboxy terminal residues of RNase A is not of lowest Gibbs free energy and highly motile despite the native like structure including 66% of the helical structure. We argue that if the conventional pair-wise interactions were to collectively account for the energy state of RNase-(1-118) the derivative should have been of lowest free energy. Thus, to explain the observations we hypothesize that some delocalized interaction exists in native RNase A to stabilize the structure. We refer to this new interaction as closed loop interaction assuming that a closed loop consisting of contacting groups would mediate delocalized interaction. Using the fragment complex system of cytochrome c developed in this Section, we wish to identify the hypothetical closed loop or loops that controls folding of cytochrome c. We have investigated hybrid complexes formed from heme- and apofragments (or apoproteins) of horse, tuna, candida and yeast iso-l-cytochrome c. The fragments of these species are found to be completely exchangeable with the following exception. Combination of tuna heme fragment (1-38)H and candida apofragment (61-109), but not that of candida heme (1-44)H and tuna apofragment (39-104), forms a complex with drastically decreased binding force. Hybrid complexes containing yeast apofragment (44-108) were not investigated. Comparing the amino acid sequences it is proposed that substitutions of leucine 9 (horse numbering) with threonine and leucine 98 with methionine are responsible for the weak binding of tuna heme-candida apofragment complex. This and other observations taken together with those previous have allowed us to assign the locations of four closed loops 1,2,3 and 4 to be above, at the right and the left sides of, and below the heme, respectively. Further, the previously found initial second order kinetic phase and the following first order phase are assigned to represent formations of closed loops 1 and 3, respectively. Loop 4 is presumed to have been added during evolution of eukaryotic cytochrome c from prokaryotic species.
